We propose to form a research collaboration in which we improve upon the screening and evaluation technologies currently used to generate affinity reagents. We intend to refine the discovery process so that it is not only cheaper and faster than currently possible, but also so that we produce reagents that are more versatile and useful than current monoclonal antibodies. This Center will involve the laboratories of Drs. Brian Kay (University of Illinois at Chicago), Andreas Pluckthun (University of Zurich), and Michael Weiner (lllumina Corporation). We will focus on three types of recombinant affinity reagents: human single-chain Fragments of variable regions (scFv), Designed Ankyrin Repeat Proteins (DARPins), and fibronectin type III (FN3) monobodies. Dr. Aled Edwards (Structural Genomics Center) will supply us with 100 targets to which we will apply our technology improvements. We will also use synthetic peptides, corresponding to the N-termini and C-termini of a test set of proteins, as targets. Our research plan consists of three strategic goals. First, through molecular engineering we will generate versatile affinity reagents that can accomplish the many standard tasks of antibodies, as well as function as biosensors and inducible inhibitors of intracellular targets. Second, we plan to improve our current selection protocols by using emulsions to make them faster, smaller, cheaper, and better. Third, in addition to our own characterization, we will distribute affinity reagents to five beta-testers for their evaluation in biological applications and receive feedback on what additional properties should be engineered into the affinity reagents. Once we have prepared and vetted high quality affinity reagents, they will be made available through the DNA repository at Arizona State University and two reagent supply companies. The overarching goal of our research will be to optimize technologies that can be applied to the entire human proteome in a versatile, powerful, cost-effective, and high-throughput manner.